Quaking phenotype influences brain lipid-related mRNA levels

Neuroscience Letters, 141 (1992)195 198 t" 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

195

NSL 08758

Quaking phenotype influences brain lipid-related mRNA levels J.W. DeWille a n d N.J. F a r m e r The Ohio Staw University, Department q]' l'i,terinar|" Pcithohiok~y, Columbus, OH 43210 ( USA

(Received 29 October 1991: Revised version received 3 April 1992; Accepted 15 April 1992)

Key words: Quaking: Myelin: Stearoyl CoA desaturase: LDL receptor: Apolipoprotein E: Gene expression

Although lipids compose almost 80% of myelin, the influenceof quaking on mRNAs encoding lipid biosyntheticenzymesand transport proteins has not been previouslyreported. Understanding the influenceof quaking on myelin-specificand lipid-relatedmRNAs will be useful in determining the mechanism of the quaking defect. StearoylCoA desaturase (SCD) catalyzesa key step in the biosynthesisof oleic acid (C 18:1, n-9), a major fany acid in myelin. SCD, LDL receptor (LDLR) and apolipoprotein E (Apo E) mRNA levelsare all reduced in neonatal quaking brains. In contrast to brain, quaking hepatic LDLR and Apo E mRNA levelsare normal. These results indicale that lipid-relatedmRNAs are reduced in neonatal quaking brain, but the quaking liver is unaffected.The quaking defect influencesgene expression in multiplecell types of glial lineage in the developingCNS.

The 'quaking' dysmyelinating mutant mouse has been extensively studied but the specific gene defect remains unknown [1, 11]. Available evidence indicates that the quaking phenotype results from a mutation localized to mouse chromosome 17 [1, 11]. The primary defect in quaking is an arrest in myelination of the central nervous system (CNS) [1, 11]. Decreased peripheral myelin content has also been described [1, 11]. The brain content of the major myelin proteins, proteolipid protein (PLP) and myelin basic protein (MBP), is dramatically reduced in quaking mice [1, 11]. PLP and MBP m R N A levels are reduced in quaking brains during the early neonatal myelinating period [1, 14, 22]. Myelin is a relatively rigid membrane system rich in phospholipids and cholesterol [7, 19]. The content of almost all classes of myelin structural lipids is decreased in brains of quaking mice [7, 11]. The activity of several key lipid biosynthetic enzymes is also reduced in quaking mouse brain [11]. The developmental regulation of lipid biosynthesis and the accumulation of structural lipids during myelination is not well understood. The fatty acid composition of myelin is largely saturated and mono-unsaturated fatty acids [3, 6]. Oleic acid (C18:1, n-9) is the major monounsaturated fatty acid in myelin, comprising 2 0 4 0 % of myelin fatty acids [3, 6, 8]. Myelin oleic acid content is about 5-fold higher than CorresT~ondence: J.W. DeWille,The Ohio State University,Department of VeterinaryPathobiology,Columbus, OH 43210, USA. Fax: (1) (614) 292-6473

brain synaptosomal, mitochondrial and microsomal oleic acid content [6]. The elevated concentration of oleic acid in myelin suggests that this fatty acid plays an important structural role in the myelin sheath. Stearoyl CoA desaturase (SCD) catalyzes the rate limiting step in the endogenous synthesis of oleic acid [12]. SCD is present in a variety of tissues including liver, brain, kidney, lung and adipose tissue [13, 20, 24]. Two SCD structural genes have been identified in rodents [13, 20, 24]. These two SCD genes share >87% amino acid sequence homology, but differ markedly in promoter sequence and tissue specific expression [13]. In adult mice, the hepatic form of SCD is inducible by diet, but the brain form of SCD appears to be constitutively expressed [13]. Brain SCD m R N A levels increase during the neonatal myelinating period, paralleling the increase in PLP and MBP M R N A levels [2]. This suggests that the expression of genes encoding enzymes involved in the synthesis of myelin structural lipids are coordinately regulated with the major myelin protein encoding MRNAS. In addition to SCD, mRNAs encoding the LDL receptor, H M G CoA reductase and H M G CoA synthase have been detected in neonatal brain [10, 15]. Apolipoprotein E (Apo E) m R N A has also been found in the developing CNS [4]. This suggests that a lipid transport system may operate in the CNS involving Apo E and cells expressing the L D L receptor [4, 21]. The expression of known 'myelin-specific' genes (i.e. PLP and MBP) must be coordinately regulated with

196

genes providing structural lipids and sterols lot myelin assembly. Our findings indicate that quaking brain SCD, LDL receptor and Apo E mRNA levels are reduced in concert with PLP and MBP mRNA levels. The quaking phenotype has no influence on hepatic lipogenic enzyme mRNA levels. Homozygous 'quaking' mice and age-matched controls were obtained from Jackson Laboratories (Bar Harbor, ME). All mice were 21-26 days of age (mean 23 days) when killed. Total RNA was isolated from brains and livers by the guanidium isothiocyanate procedure followed by cesium chloride centrifugation [23]. Tissues from 2-4 mice were pooled for each experiment. Each experiment was replicated 2-3 times. Poly A + RNA was isolated from total RNA by oligo-dT cellulose chromatography [23]. RNA was quantitated by absorbance at 260 nm and purity assessed by determining the ratio of absorbance at 260 and 280 nm (A260/280) [23]. Ten micrograms of Poly A ÷ RNA was electrophoresed through a 1.2% formaldehyde-agarose gel and transferred to a nylon filter. Human PLP, MBP, LDL receptor and apo E and chicken fl-actin cDNAs were obtained from American Type Culture Collection [18]. Mouse brain- and liver-specific Stearoyl CoA desaturase (SCD) partial cDNA inserts were provided by Dr. M.D. Lane, Johns Hopkins University. All cDNA inserts were excised from plasmid constructs and the band-isolated fragment was labelled by the random primer method [5]. Hybridization was carried out for 18-24 h at 42°C in a rotating hybridization incubator in solutions containing 50% formamide, sodium chloride/sodium phosphate/ EDTA buffer (pH=7.4) (SSPE), Denhardt's solution, 0.5% SDS, 4% dextran sulfate and 100/lg/ml denatured, sheared salmon sperm DNA. Filters were washed with two changes of 2xSSPE/0.1% SDS at room temperature for 30 min followed by 1 3 additional washes with 0.1xSSPE/0.1% SDS at room temperature to 50°C for 15 60 rain until background counts were negligible relative to hybridization signals. Filters were exposed to Xray film in the presence of an intensifying screen at -70°C The mean age of quaking and control mice used in this study was identical (23 days old). Mean wet weights of quaking brains were slightly less than controls (404 mg vs. 441 mg). Quaking mouse body and liver weights were also slightly lower than age-matched controls. Histological examination indicated hypomethylation of neonatal quaking brains, but no additional pathology was observed (data not shown). To insure that equal amounts of quaking and control RNA were loaded onto gels for Northern analysis RNA was quantitated by absorbance at 260 nm immediately prior to gel loading and hybridized to a non-specific (fl-actin) probe at the end of the

experiment. The quantity and integrity of mRNA loaded was also checked by ethidium bromide and Methylene blue staining [23]. Quaking PLP mRNA levels were significantly decreased compared with controls (Fig. IA, lane 1: quaking, lane 2: control). Using a full-length human PLP cDNA probe we detected PLP mRNA bands of approximately 3.2 and 2.4 kb, indicative of multiple polyadenylation sites [I 7]. Both these findings are consistent with previous comparisons of PLP mRNA levels in quaking and normal mice [14]. In addition to PLR the quaking phenotype also influences MBP mRNA levels [1, 14, 22]. Quaking MBP mRNA levels were reduced compared with controls (Fig. 1B, lane l: quaking, lane 2: normal). SCD mRNA levels increase in a developmentally regulated manner in concert with PLP and MBP [2]. Both PLP and MBP mRNA levels are reduced in the brains of neonatal quaking mice [1, 14, 22]. The influence of the quaking phenotype on neonatal SCD mRNA levels was determined. Consistent with the results for PLP and MBR SCD mRNA levels are significantly decreased in brains of neonatal quaking mice (Fig. 1C, lane 1: quaking, lane 2: control). The myelin sheath is approximately 30% cholesterol [7, 11]. LDL receptor, HMG CoA reductase and HMG CoA synthase mRNAs are present in neonatal brain, suggesting that both exogenous uptake and endogenous synthesis may function during the neonatal myetinating period [4, 10, 15, 21]. Quaking brain LDL receptor mRNA levels are reduced compared to controls (Fig. 2A,B, lane 1: quaking, lane 2: control). Quaking phenotype has no effect on brain fl-actin mRNA levels (Fig. 2B) or liver LDL receptor mRNA levels (data not shown). Although liver is the major site of apolipoprotein syn-

A.

1

PLP

2

1

2

I

2

Fig. I. Influence of quaking phenotype on: (A) proteolipid protein (PLP), (B) myelin basic protein (MBP), and (C) stearoyl C o A desaturase (SCD) m R N A levels.Ten micrograms of poly A ~ R N A isolated from brains of 23 day old quaking (lane I) and control mice (lane 2) were analyzed by Northern blot. Blots were probed with riP-labelled eDNA inserts from: human PLP (A), human MBP (B) and mouse SCD (C). Molecular size markers 28S (upper) and 18S (lower) are indicated by hash marks ( ) .

197

A)

LDL Receptor

Apo E 18S-

288-

., o~~Si~ ~

B)

Actin 1

2 Brain

3

4 Liver

Fig. 3. Influence of quaking phenotype on brain and liver Apo E mRNA levels. Ten micrograms of poly A" RNA isolated from 23 day old quaking brain (lane 1) and liver (lane 3) were compared with agematched control mouse brain (lane 2) and liver (lane 4). Poly A + RNA was analyzed by Northern blot probed with '-'P-labelled human Apo E eDNA insert. 18S-

1

2

Fig. 2. Influence of quaking phenotype on brain (A) LDL receptor and (B) Actin mRNA levels. Ten micrograms of poly A- RNA isolated from brains of 23 day old quaking (lane 1) and control (lane 2) mice were analyzed by Northern blot. Blots were probed with '2P-labelled human LDL receptor eDNA insert (A) and chicken fl-actin cDNA insert (B).

thesis, significant quantities of Apo E are present in the developing CNS [4, 21]. Apo E in the CNS is synthesized by astrocytes, which actively metabolize lipids [4, 9, 2l]. Quaking brain Apo E m R N A levels are decreased compared with controls (Fig. 3, lane 1: quaking, lane 2: control). In contrast, quaking and control liver Apo E m R N A levels are equal (Fig. 3, lane 3: quaking, lane 4: control). Although lipids comprise almost 80% of myelin [7, 11] the influence of the quaking phenotype on mRNAs encoding lipid biosynthetic enzymes and transport proteins has not been characterized. Identifying genes that are coordinately regulated in concert with the 'myelin-specific' (PLP and MBP) genes provides a broader base from which to characterize the mechanism of the quaking phenotype. Quaking is associated with reduced PLP m R N A levels during the neonatal myelinating period [14]. Although PLP m R N A levels are reduced, processing of the PLP m R N A is normal in quaking brain [14]. The influence of quaking on MBP m R N A levels is variable [1, 14, 22].

Konat et al., found that MBP mRNA levels in 18 day old quaking brain are reduced, but MBP mRNA levels in 27 day old quaking brain are almost normal [14]. Our results show that at 23 days of age, MBP m R N A levels in brains of quaking mice is reduced compared with control brains. This indicates that quaking MBP mRNA levels increase late in the neonatal myelinating period. We recently showed that SCD m R N A levels increase in concert with PLP and MBP and all three are developmentally regulated in neonatal mouse brain [2]. Although SCD does not encode a myelin constituent, SCD encodes the rate limiting step in the synthesis of oleic acid, a key myelin structural component [12, 13]. These data are consistent with the hypothesis that PLP, MBP and SCD m R N A levels are coordinately regulated during myelination. Hepatic SCD gene expression is regulated in an entirely different manner, unrelated to brain SCD gene expression [13, 20, 24]. Hepatic SCD mRNA levels are not altered in quaking mice (data not shown). The LDL receptor provides a mechanism for cellular uptake of exogenous cholesterol [10, 15, 21]. This mechanism may function in neonatal brain, particularly during the neonatal myelinating period [10, 15]. LDL receptor m R N A levels are reduced in neonatal quaking brain. Analysis of the endogenous cholesterol biosynthetic pathway is in progress to determine its role in cholesterol accumulation in normal and quaking mouse brain. Apo E binds with high affinity to the LDL receptor [4, 16, 21]. Reduced quaking brain Apo E m R N A levels indicate that the postulated neural Apo E lipid transport system is impaired due to the absence of both the LDL receptor

198

and its high affinity ligand, Apo E. It is noteworthy that Apo E is produced in astrocytes, the non-myetinating cells of glial lineage [4, 16, 21]. Astrocytic abnormalities have also been reported in the 'jimpy' dysmyelinating mutant [1]. This suggests that both jimpy and quaking contain defects in multiple cells of glial lineage; i.e. both astrocytes and oligodendrocytes. The quaking phenotype does not impair hepatic LDL receptor or Apo E mRNA levels. The pattern of dysmyelination in the quaking CNS exhibits a caudal-rostral gradient [1], suggesting an arrest in oligodendrocyte differentiation [1]. Current experiments are aimed at investigating the developmental regulation of myelin-specific genes and determining myelin-specific transcriptional control factors. This work was supported by an Ohio State University Seed Grant. l Campagnoni, A.T. and Macklin. W.B., Cellular and molecular aspects of myelin protein gene expression, Mol. Neurobiol., 2 (1988) 41 89. 2 DeWille, J.W. and Farmer, S.F., Dietary essential fatty acids influence neonatal brain lipogenic mRNA levels, Dev. Neurosci,, 14 (1992) 61 68. 3 Divakaran, P., Pavlina, T., Johnson, R.C., Cotter, R., Madsen. D. and Wiggins, R., Dietary supplementation of undernourished rats with soy or safflower oil: effects on myelin polyunsaturated fatty acids, Met. Brain Res., 1 (1986) 157164. 4 Elshourbagy, N.A., Liao, W.S., Mahley, R.W. and Taylor, J.M., Apotipoprotein E mRNA is abundant in the brain and adrenals, as well as the liver, and is present in other peripheral tissues of rats and marmosets, Proc. Natl. Acad. Sci. USA, 82 (1985) 203 207. 5 Fineberg, A.P. and Vogelstein, B., A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Anal, Biochem., 132 (1983) (~ 13. 6 Galli, C., Trezeciak, H.I. and Paoletti, R., Effects of essential fatty acid deficiency on myelin and various subcellular structures in rat brain, J. Neurochem., 19 (1972) 1863- 1867. 7 Ganser, A.L., Kerner, A.-L., Brown, B.J., Davisson, M.T. and Kirschner, D.A., A survey of neurological mutant mice I. Lipid composition of myelinated tissue in known myelin mutants, Dev. Neurosci., 10 (1988) 99-122. 8 Gustavsson, L. and Ailing, C., Effects of chronic ethanol exposure on laity acids of rat brain glycerophospholipids, Alchohol, 6 (1989) 13(,, 146. 9 Hertting, G. and Seegi, A., Formation and function of eicosanoids in the central nervous system, Ann. N.Y. Acad. Sci., 559 (1989) 84 99.

10 Hofmann, S.L., Russell, I).W.. Gotdstem, J.L. and Brown, M.S., mRNA for low density lipoprotein receptor in brain and spinal cord of immature and mature rabbits, Proc. Natl. Acad. Sci, USA. 8-I (1987)6312 6316. 11 Hogan, E.L. and Greenfield, S., Animal models of genetic disorders of myelin. In R Morrell (Ed.), Myelin, Plenum, New York. 1984, pp. 489-534. 12 Jeffcoat, R. and James, A.T., The regulation of desaturation anti elongation of fatty acids in mammals. In S. Numa (Ed.), Fatty acid metabolism and its regulation, Elsevier. New York, 1984, pp. 85 112. 13 Kaestner, K.H., Ntambi, J.M., Kelly, T.J. and Lane, M.D., Differentiation-induced gene expression in 3T3-Lt preadipocytcs, J. Biol. Chem., 264 (1989) 14755: 14761. 14 Konat, G., Trojanowska, M., Gantt, G. and Hogan, E.L., Expression of myelin protein genes in quaking mouse brain. J. Neurosci. Res., 20 (1988) 19 22. 15 Levin, M.S,, Pitt, A.J,A., Schwartz, A.L., Edwards, P.A. and Gordon, J.l,, Developmental changes in the expression o1 genes involved in cholesterol biosynthesis and lipid transport in human and rat fetal and neonatal livers, Biochem. Biophys. Acta, 1003 (1989) 293-300, 16 Mahley, R.W., Apolipoprotein E: cholesterol transport protein with expanding role in cell biology, Science, 240 (1988) 622 -630. 17 Milner, R.J., Lai, C., Nave, K.-A., Lenoir, D., Ogata, J. and Sutcliffe, J,G., Nucleotide sequences of two mRNAs for rat brain myelin proteolipid protein, Cell, 42 (1985) 931 939. 18 Nierman, W.C. and Maglott, D.R., American Type Culture Collection, NIH Repository of Human and Mouse DNA Probes and Libraries, 1989. 19 Norton, W.T. and Cammer, W., isolation and characterization of myelin. In P. Morrell (Ed.), Myelin, Plenum, New York, 1984, pp. 147 195. 20 Ntambi, J.M., Buhrow, S.A., Kaestner, K.H., Christy, R.J., Sibley, E., Kelly, T.J. and Lane, M.D., Differentiation-induced gene expression in 3T3-L I preadipocytes. J. Biol. Chem., 263 (1988) 17291 17300. 21 Pitas, R.E., Boyles, J.K., Lee, S,H., Hui, D. and Weisgraber, K.H_ Lipoproteins and their receptors in the central nervous system, Characterization of the lipoproteins in the cerebralspinal fluid and identification of apolipoprotein B,E(LDL) receptors in the brain, J, Biol. Chem., 262 (1987) 14352 14360. 22 Roth, H.J., Hunkeler, M.J. and Campagnoni, A.T., Expression of myelin basic protein genes in several dysmyelinating mouse mutants during early postnatal brain development, J. Neurochem., 45 (1985) 572 580. 23 Sambrook, J., Fritsch, E.F. and Maniatis, T., Extraction, purification, and analysis of messenger RNA from eukaryotic ceils. In: Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989, pp. 7.2 7.87. 24 Thiede, M.A. and Strittmatter, P., The induction and characterization of rat liver stearyl-CoA desaturase mRNA, J. Biol. Chem., 260 (1985) 14459 14463.